1. Field of the Invention
The present invention relates to an oligonucleotide and a remedy for treating immune-mediated disorders, using the oligonucleotide. The immune-mediated disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation of host's immune system by autoantigens, microbes and Toll-like receptor (TLR)-mediated disease.
2. Description of the Related Art
The present invention provides an oligonucleotide which has a nucleotide sequence of 5′-cctcctcctcctcctcctcctcct-3′ (SEQ ID NO: 1) and a remedy for treating and/or preventing immune-mediated disorder, using the oligonucleotide.
The immune system protects human body from bacterial, parasitic, fungal, viral infections and from the growth of tumor cells. However, the immune response can sometimes be unwanted and cause immune-mediated disorder. The disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation of host's immune system by microbes and Toll-like receptor (TLR)-mediated disease.
The autoimmune diseases results from an adaptive immune response and innate immune response or both against endogenous and/or exogenous antigens. Foreign substances, derived from bacteria, parasites, fungi or viruses, may mimic self-proteins and stimulate the immune system to launch responses to a self-cell and tissue, resulting in the diseases including but not limited to systemic lupus erythematosus □SLE□ and rheumatoid arthritis. The graft rejection is a consequence of organ or tissue transplantation caused by the immune response in the transplant recipient (host) to the transplanted organ/tissue. When a subject is transplanted with grafts including kidney, pancrea, heart, lung, bone marrow, cornea and skin, the subject can launch an immune response (rejection) against the grafts. Hypersensitivity is an inappropriate immune response that has deleterious effects, resulting in significant tissue damage or even death. The hypersensitivity is divided into four types (e.g. Types I, II, II and IV. Disease associated with the over-stimulation of host's immune system by microbes is triggered by the infection of viruses such as flu viruses and other microbes. In the case of flu virus and Gram-negative bacterial infection, an excessive immune response to the invaders appears to be a fatal factor in patients. The response is characterized by the overproduction of cytokines. Studies of septic shock syndrome demonstrate that over production/aberrant production of cytokines can lead to rapid mortality due to cytokine-mediated lethal shock (Slifka M K, et al. J Mol Med. 2000; 78(2):74-80). Septic shock following gram-negative infection is a leading cause of mortality in critically ill patients. The exaggerated production of cytokines is known to contribute to sepsis characterized by cytokine-mediated lethal shock (Espat N J, et al. J Surg Res. 1995 July; 59 (1):153-8). Multiple organ dysfunction syndromes (MODS) are a major cause of morbidity and mortality in severe sepsis and shock. Cytokine-mediated lethal shock resulted from over-production of host cytokines is considered a main mechanism leading to MODS (Wang H, et al. Am J Emerg Med. 2008 July; 26 (6):711-5). Toll-like receptor (TLR)-mediated disease is a disorder caused by the activation of Toll like receptors (TLRs). TLRs are a family of receptors that recognize microbe derived molecular structures (pathogen-associated molecular patterns or PAMPs). TLR expressing immune cells are activated upon binding of PAMPs. TLRs recognize a range of pathogen-derived products and activated. Lipopolysaccharide (LPS) of bacteria recognized by TLR4, lipotechoic acid and diacylated lipopeptides by TLR2-TLR6 dimmer, triacylated lipopeptides by TLR2-TLR1 dimmer, CpG containing oligonucleotide (CpG ODN) synthesized or derived from either viruses or bacteria by TLR9, bacterial flagellin by TLR5, zymosan by TLR2-TLR6 dimmer, F protein from respiratory syncytial virus (RSV) by TLR4, viral-derived double-stranded RNA (dsRNA) and poly I:C, a synthetic analog of dsRNA by TLR3; viral DNA by TLR9, single-stranded viral RNA (VSV and flu virus) by TLR7 and TLR8 (Foo Y. Liew, et al. Nature Reviews Immunology. Vol 5, June 2005, 446-458). In recent years, TLR activation has been connected to the pathogenesis of some of diseases including sepsis, dilated cardiomyopathy, diabetes, experimental autoimmune encephalomyelitis, systemic lupus erythematosus, atherosclerosis, asthma, chronic obstructive pulmonary disease and organ failure (Foo Y. Liew, et al. Nature Review Immunology, Vol 5, 2005, 446-458). Activation of TLR9 by self DNA play an important role in the development of autoimmune diseases such as SLE (Christensen S R, et al. Immunity 2006; 25:417-28) and rheumatoid arthritis (Leadbetter E A, et al. Nature 2002; 416:603-7; Boule M W, et al. J Exp Med 2004; 199:1631-40). Moreover, the elevated production of interferons (IFNs) resulted from TLR-9 activation has been reported to contribute to the develop of systemic lupus erythematosus (Barrat F J, et al. J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102:9258-63).
In this invention, we disclose an oligonucleotide with a nucleotide sequence of 5′-cctcctcctcctcctcctcctcct-3′ (SEQ ID NO: 1) that inhibits proliferation of human PBMC activated by TLR9 agonist, inhibits interferon production from human PBMC induced by TLR9 agonist, HSV-1, flu virus and serum from SLE patients, and rescues the mice from cytokine induced shock. Therefore, this oligonucleotide is useful as a remedy for the treatment immune-mediated disorders.
The oligonucleotides of the invention inhibits TLR9 activation. It has been documented that TLR9 agonist activates both innate and adaptive immune response (Arthur M. Krieg. Nature Reviews Drug Discovery, Vol 5. June 2006, 471-484). CpG containing oligonucleotides (CpG ODN) is a TLR9 agonist [D. M. Klinman, Nat. Rev., Immunol. 4 (2004) 249-258]. The oligonucleotides of the invention inhibits the proliferation and interferon production of human PBMC stimulated by CpG ODN, indicating that the oligonucleotide of the invention can be used as a remedy for the treatment of diseases related to TLR9 activation. Because TLR9 activation has been reported to contribute to the development of SLE (Barrat F J, et al. J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102:9258-63; Christensen S R, et al. Immunity 2006; 25:417-28) and rheumatoid arthritis (Leadbetter E A, et al. Nature 2002; 416:603-7; Boule M W, et al. J Exp Med 2004; 199:1631-40), the oligonucleotide of the invention can be used as a remedy for the treatment of SLE and rheumatoid arthritis by inhibiting the TLR9 activation.
The oligonucleotide of the invention inhibits the interferon production from human PBMC induced by TLR9 agonist, HSV-1, flu virus and serum from SLE patients. Because the elevated production of interferons has been reported to contribute to the development of SLE (Barrat F J, et al. J Exp Med 2005; 202:1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102:9258-63), the oligonucleotide of the invention can be used as a remedy for the treatment of SLE by inhibiting IFN production.
The oligonucleotides of the invention inhibits the interferon production from human PBMC induced by flu virus (PR8). Since influenza virus has been documented to be able to activate TLR7 and TLR8 (Wang J P, et al. Blood. 2008 Jun. 10. [Epub ahead of print]), the oligonucleotide of the invention can be used as a remedy for the treatment of Toll-like receptor (TLR)-mediated disease by inhibiting TLR7 or TLR8.
The oligonucleotides of the invention inhibits the interferon production from human PBMC induced by HSV-1. Since HSV-1 has been documented to activate TLR9 (Hubertus Hochrein et al. PNAS, 101, 11416-11421), the oligonucleotide of the invention can be used as a remedy for the treatment of Toll-like receptor (TLR)-mediated diseases including but not limited to SLE by inhibiting the activation of TLR9.
To study in vivo activity of the oligonucleotide of the invention, a mouse model of cytokine-mediated lethal shock was used. The mouse model was created by injecting CpG ODN into the D-galactosamine (D-Gal) presensentised mice. After being crated, the model mice died within 12 to 24 h. Analyses of plasma cytokines revealed over-production of tumor necrosis factor (TNF) alpha and interleukin-12 (IL-12) and gamma interferon (IFN-gamma) (Marshall A J, et al. Infect Immun. 1998 April; 66(4):1325-33; Peter M, Bode K, et al. Immunology. 2008 January; 123(1):118-28). By using the model, we demonstrate that the oligonucleotide of the invention can rescue mice from cytokine-mediated lethal shock. Because the cytokine-mediated lethal shock contributes to the septic shock (Slifka M K, et al. J Mol. Med. 2000; 78(2):74-80; Espat N J, et al. J Surg Res. 1995 July; 59(1):153-8) and multiple organ dysfunction syndromes (MODS) (Wang H, et al. Am J Emerg Med. 2008 July; 26(6):711-5), the oligonucleotide of the invention can be used as a remedy for the treatment of sepsis and MOGS by rescuing the host from cytokine-mediated lethal shock.